Fig. 1

Tg-induced cell death depends on DR5 and caspase-8, and partially requires FADD and Fas, but not DR4 or TRADD in LNCaP cells. a LNCaP cells were transfected for 2 d with the indicated siRNAs (siCtrl = non-targeting control siRNA, siCasp8 = siCaspase-8). Subsequently, cells were treated with 100 nM Thapsigargin (Tg) or 0.01% DMSO vehicle control (“DMSO”, also transfected with siCtrl) in the additional presence of 2.5 μg/ml propidium iodide to stain dead cells. Cell death was monitored and quantified with the IncuCyte ZOOM as described in Materials and Methods, and displayed as relative values normalized to those obtained in the siCtrl+Tg condition after 48 h of treatment (mean value set to 1). b LNCaP cells were transfected and treated as in (a). After 30 h of treatment with Tg or DMSO (also transfected with siCtrl), whole cell lysates were prepared and subjected to western blotting with the indicated antibodies; p55/53 Casp8 = procaspase-8, Casp3 = caspase-3 (only cleaved caspase-3 bands are shown), cl-PARP = cleaved PARP. The positions of molecular weight markers are indicated to the left, and nonspecific bands marked with an asterisk. One representative blot out of 4 independent experiments. c and d Quantifications of the protein levels of cleaved Caspase-3 (c) and cleaved PARP (d), normalized to the tubulin loading control and then to the Tg + siCtrl condition (mean value set to 1). For (a, c, and d): Mean ± SEM from 4 independent experiments. Dots represent individual values, with a separate color for each experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ns; not significant, One-way ANOVA compared to the Tg + siCtrl condition